GSD-Ib patients manifest neutrophil dysfunction of unknown etiology. Previously, we show that G6PT interacts with the enzyme G6Pase-beta to regulate the availability of G6P/glucose in neutrophils. A deficiency in G6Pase-beta activity in neutrophils impairs both their energy homeostasis and functionality. We now show that human G6PT-deficient neutrophils are similarly impaired. Their energy impairment is characterized by decreased glucose uptake and reduced levels of intracellular G6P, lactate, ATP, and NADPH, while functional impairment is reflected in neutrophil respiratory burst, chemotaxis, and calcium mobilization. We show that the expression and membrane translocation of the NADPH oxidase subunit p47phox is downregulated in G6PT-deficient neutrophils, explaining why respiratory burst activity is impaired. We further show that the hypoxia-inducible factor-1alpha (HIF-1alpha)/peroxisome proliferators-activated receptor-gamma (PPAR-gamma) pathway that directly impacts neutrophil respiratory burst, chemotaxis, and calcium mobilization is activated in G6PT-deficient neutrophils. Finally, we show that exposing human G6PT-deficient neutrophils to a PPAR-gamma antagonist improves their function. Taken together, our results demonstrate that the underlying cause of neutrophil dysfunction in GSD-Ib arises from impaired neutrophil energy homeostasis and activation of the HIF-1alpha/PPAR-gamma pathway. G6Pase-beta is an enzyme catalyzing the hydrolysis of G6P to glucose. To date, 33 separate G6Pase-beta mutations have been identified in GSD-Irs patients but only the p.R253H and p.G260R missense mutations have been characterized functionally for pathogenicity. We now functionally characterized 16 of the 19 known missense mutations using a sensitive assay, based on a recombinant adenoviral vector-mediated expression system, to demonstrate pathogenicity. A database of residual enzymatic activity retained by the G6Pase-beta mutations will serve as a reference for evaluating genotype-phenotype relationships. We have shown that gene therapy mediated by rAAV8-G6PC, a recombinant AAV vector expressing human G6Pase-alpha directed by the human G6PC promoter/enhancer normalizes blood glucose homeostasis in G6pc-/- mice for 70-90 weeks. The treated G6pc-/- mice expressing 3-63% of normal hepatic G6Pase-alpha activity (AAV mice) produce endogenous hepatic glucose levels 61-68% of wild-type littermates, have a leaner phenotype, and exhibit fasting blood insulin levels more typical of young adult mice. We now show that unlike wild-type mice, the lean AAV mice have increased caloric intake and do not develop age-related obesity or insulin resistance. Pathway analysis shows that signaling by ChREBP that improves glucose tolerance and insulin signaling is activated in AAV mice. In addition, several longevity factors in the calorie restriction pathway, including: the NADH shuttle systems; NAD+ concentrations; and the AMPK/SIRT1/PGC-1alpha pathway are up-regulated in the livers of AAV mice. Our study suggests that full restoration of normal G6Pase-alpha activity will not be required to confer significant therapeutic benefits in GSD-Ia by gene therapy. The finding that a moderate reduction of hepatic G6Pase-alpha activity in mice may promote a leaner phenotype and prevent the development of age-related decrease in insulin sensitivity lends weight to the suggestion that G6Pase-alpha constitutes a potential pharmaceutical target for treating type 2 diabetes. GSD-Ia is characterized by impaired glucose homeostasis and long-term complication of hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC). Using G6pc-/- mice, we have shown that systemic administration of rAAV8-G6PC delivers G6Pase-alpha to the liver and corrects hepatic G6Pase-alpha deficiency. We further show that 70-90 week-old rAAV-G6PC-treated G6pc-/- mice, expressing &#8805; 3% of normal hepatic G6Pase-alpha activity maintain glucose homeostasis and show no evidence of HCA/HCC. We now generated rAAV-co-G6PC, a rAAV8 vector expressing a codon-optimized (co) human G6Pase-alpha (co-G6PC) directed by the human G6PC promoter/enhancer and investigated the impact of codon optimization strategies on translation efficiency. In a 66-80-week study, we showed that the rAAV-co-G6PC vector is more efficacious than the rAAV-G6PC vector in directing hepatic G6Pase-alpha expression. As expected, the treated mice expressing &#8805; 3% of normal hepatic G6Pase-alpha activity display normal metabolic profiles, maintain normoglycemia over a 24-hour fast, show no evidence of HCA/HCC, and are protected against age-related obesity and insulin resistance. Among the twenty-six 66/80-week-old rAAV8-G6PC- and rAAV8-co-G6PC-treated G6pc-/- mice, twelve mice expressed hepatic G6Pase-alpha activity &#8804; 3% of normal hepatic G6Pase-alpha activity. Three of these mice expressing 0.9% to 1.3% of normal hepatic G6Pase-alpha activity developed HCA/HCC, establishing the threshold of transgene expression required to prevent HCA/HCC. We further show that ChREBP signaling that improves glucose tolerance and insulin sensitivity is activated in all rAAV-treated mice. However, activation of ChREBP signaling is significantly attenuated in the HCC nodules. We have shown that systemic administration of rAAV-CBA-G6PT, a rAAV8 vector expressing human G6PT directed by the hybrid chicken &#61538;-actin (CBA) promoter/CMV enhancer, delivered the G6PT transgene to the liver and normalized metabolic abnormalities in murine GSD-Ib. However, the 5 transduced GSD-Ib mice that lived to age 52-70 weeks expressed only <4% of wild-type hepatic G6PT activity and two mice developed HCA with one undergoing malignant transformation. Studies have shown that the choice of transgene promoter not only impacts targeting efficiency and tissue-specific expression, but also the level of immune response or tolerance to the therapy. We therefore examine the safety and efficacy of rAAV8-G6PT, a rAAV8 vector expressing human G6PT directed by the tissue-specific human G6PC promoter/enhancer. Among the fifteen 60-78 week-old rAAV-treated G6pt-/- mice expressing 2-62% of wild-type hepatic G6PT activity, only one mouse expressing 6% of normal hepatic G6PT activity developed HCA. The rAAV-treated mice, including the HCA-bearing mouse displayed normal hepatic fat storage, normal glucose tolerance profiles, and maintained normoglycemia over a 24-hour fast. The rAAV-treated mice also exhibit a leaner phenotype and are protected against age-related insulin resistance. We further show that activation of hepatic ChREBP signaling that improves glucose tolerance and insulin sensitivity is one mechanism that protects the rAAV-GPE-G6PT-treated G6pt-/- mice against age-related obesity and insulin resistance.